Phase I: High throughput fabrication of fractal cell culture substrates

目前，还没有细胞培养技术能够高保真地培养关键的肾脏细胞：足细胞。单层培养不能概括其分枝表型。我们开发了一种全新的方法：通过在细胞培养基质上忠实地重建天然肾小球的分形形态线索，我们微工程创造了一个足细胞展示其分支形态和与体内足细胞相似的基因表达的环境。这种改进的细胞培养在新的化合物测试和冠状病毒感染研究中比传统的单层培养更优越。这项提议的主要目标是开发一种用于生产具有分形地形线索的商业井板的高通量制造工艺，从目前需要20小时生产一块2x2平方厘米基板的手工工艺，到每块平板需要15分钟的工艺。在目标1中，我们将通过热压花和注塑扩大生产，以创造商业24孔板和96孔板。在目标2中，我们将制定这些平板的灭菌方案，在目标3中，我们将验证它们的生物兼容性。为了达到所需的生产能力并制造出非吸水性设备，我们需要克服从聚二甲基硅氧烷实验室原型插件到玻璃上层压的非吸水性热塑性弹性体的主要工程挑战。我们的主要客户将是制药公司，例如填写了订单的Regeneron，以及需要高保真足细胞培养的学术实验室。我们的合作者夸金博士、巴鲁阿博士和罗杰斯博士是领先的肾脏研究人员，他们渴望在他们的实验室中测试新的足细胞培养皿。新的制造工艺也可以用于制造其他细胞的分形板，例如脂肪细胞和肺细胞。我们首选的模式是将平板授权给现有的制造和分销生命科学研究创新工具的老牌公司，例如提供支持信的公司-干细胞技术公司和UFluidx公司。这些平板还将通过我们的团队成员将致力于的一个新的初创公司，使我们能够发现治疗肾脏疾病的新化合物。

Currently, there are no cell culture techniques that enable high fidelity cultivation of the key kidney cells: podocytes. Monolayer culture fails to recapitulate their branching phenotype. We have developed a completely new approach: by faithfully recreating fractal topographical cues of the native kidney glomerulus on cell culture substrates, we microengineered an environment where podocytes exhibit their branching morphology and gene expression similar to the podocytes in vivo. Such improved cell culture was superior in new compound testing and coronavirus infection studies compared to the conventional monolayers. The main goal of this proposal is to develop a high throughput manufacturing process for production of commercial well plates with fractal topographical cues, from the current manual process that takes >20hr to produce one 2x2cm2 substrate, to a process that takes 15min per plate. In Aim 1, we will scale-up production by hot embossing and injection molding to create commercial 24- and 96-well plates. In Aim 2, we will develop sterilization protocol for these plates and in Aim 3, we will validate their biocompatibility. To reach the desired production throughput and create non-absorbent devices, we need to overcome the main engineering challenge of scaling from polydimethylsiloxane lab prototype inserts to non-absorbent thermoplastic elastomer laminated on glass. Our main customers will be pharmaceutical companies, e.g. Regeneron that filled an order form, and academic laboratories who have a need for high fidelity culture of podocytes. Our collaborators, Drs. Quaggin, Barua and Rogers, are leading kidney researchers, eager to test the new plates for podocyte cultivation in their laboratories. The new manufacturing process can also be used to fabricate fractal plates for other cells e.g. adipocytes and lung cells. Our preferred model is licensing the plates to an existing established company that manufactures and distributes innovative tools for life science research, such as companies that provided support letters-Stem Cell Technologies and uFluidix. The plates will also enable discovery of new compounds for kidney disease, through a new start-up that our team members will work on.